The ultimate goal of this project is to elucidate the molecular and cellular regulatory mechanisms of miR-140 in colon cancer. Human colon cancer is highly heterogeneous, and systemic drug treatment is almost never able to cure individuals with late stage tumors. One major reason for the failure of chemotherapy is the resistant cancer stem cell population: current chemotherapeutics target rapidly dividing cancer cells, but cancer stem cells divide slowly, and thus are relatively resistant to cytotoxic systemic therapies. Our recent studies showed that several non-coding miRNAs are involved in the resistance mechanism in colon cancer stem cells. In particular, we demonstrated that the cell proliferation of differentiated colon cancer cells was significantly reduced by over- expressing miR-140. As a result, these cells became more resistant to 5-fluorouracil (5-FU) or methotrexate (MTX) treatment. We further revealed that the expression of miR-140 was elevated in a small population of CD133+HICD44+HI colon cancer stem-like cells. These cells are highly resistant to 5-FU treatment, and by blocking miR-140 activity using anti-miR140 oligonucleotides, we were able to increase chemosensitivity to 5-FU (1). We also discovered that one of the key targets of miR-140 is histone deacetylase 4 (HDAC4), implying that miR-140 could potentially modulate transcriptional activation of genes involved in cell cycle control through HDAC4. Given the potential of this new paradigm whereby miRNAs, acting post-transcriptionally, can impact pathways leading to cancer growth and altered chemosensitivity, to offer new options for treatment, it is essential to follow up and extend our findings: We therefore propose three specific aims: (1) To investigate the mechanisms of miR-140 in contributing to proliferation, differentiation and chemoresistance in CD133+HICD44+HI colon cancer stem cells in vitro and in vivo; (2) To investigate the impact of miR-140 on colon cancer metastasis; and (3) To define the molecular targets and interaction networks impacted by miR-140, taking advantage of a powerful new approach, TrIP-Seq, to identify miRNA regulation at the translational level, even in small stem cell populations. This proposed project will provide the molecular basis for developing miR-140-based therapeutic strategies to overcome chemoresistance in colon cancer stem cells and to improve patient survival.